BiP Binding Research Articles

Asparagine-Linked Glycosylation at the NH2-Terminus Does Not Influence Secretion or Function of Coagulation Factors V and VIII

AbstractAbstract 2220The commercial production of recombinant factor VIII (rFVIII) is 2 to 3 orders of magnitude lower than that of comparably sized proteins in heterologous mammalian cell expression systems. One of the major contributing factors to its very low expression levels is protein misfolding and chaperone mediated retention in the endoplasmic reticulum (ER). Glucose regulated protein 78, also known as BiP, a central player in ER homeostasis, plays an important role in retention of FVIII in the ER. The secretion of Factor V (FV), a coagulation factor homologue of FVIII, is much more efficient (∼10- to 15-fold higher) in mammalian cells. Although the proteins share an identical domain structure, with close to 40% homology in their A and C domains, unlike with FVIII, BiP does not bind to and retain FV in the ER. Successful bioengineering strategies to improve the secretion of FVIII have been rationally informed by detailed study of ER chaperone interactions. A previous report indicated that the presence of multiple asparagine (N)-linked glycans within the first 50 amino acid residues of the NH2-terminus of some glycoproteins steers the nascent polypeptide away from BiP and towards the calnexin-calreticulin lectin protein folding machinery, resulting in more efficient folding and secretion of the protein (Molinari & Helenius, Science 2000). We hypothesized that the lack of BiP binding and the more efficient secretion of FV over FVIII could be the result of the presence of two N-linked glycans at positions 23 and 27 in FV, whereas FVIII has a single N-linked glycan at position 41. We investigated the role of N-linked glycans at the NH2-termini in the secretion and function of FV and FVIII. Single and double glycosylation mutants of FV were created by replacing Asn residues with Gln residues at positions 23 and 27. Similarly, Asn at position 41 of FVIII was mutated to Gln. The mutants were transiently transfected in COS-1 and CHO cells and their secretion and function were analyzed and compared to that of the respective wild type (WT) proteins. Antigen and activity assays revealed that the secretion and function of single and double glycosylation mutants of FV were no different from the WT protein indicating that the two N-linked glycans at the NH2–terminus did not contribute to the secretion or function of FV. Similarly, the FVIII glycosylation mutant did not display a significant reduction in secretion or function. We next tested if increasing the density of N-linked glycosylation at the NH2–terminus of FVIII might help improve folding and secretion. Two additional N-linked glycosylation sequons were introduced at positions 17 and 47 of FVIII by mutating M17N and K47N. Single and double glycosylation mutants were analyzed by transfections in COS-1 and CHO cells and compared to the WT protein. While the M17N glycosylation mutant was quite similar to the WT protein, the K47N mutant displayed a significant reduction in secretion. However, K47 is located adjacent to K48, a residue which has been shown to be the site of known hemophilia A missense mutations. The secretion of the M17N/K47N double mutant was reduced even further to almost half of the WT protein in cell media. We conclude that, unlike some other glycoprotein-ER chaperone interactions, early N-linked oligosaccharides at the NH2–termini of FV and FVIII do not contribute significantly to the secretion and function of either protein. Previous structural comparisons between FV and FVIII have revealed that a stretch of hydrophobic amino acids within the FVIII A1 domain may contribute to BiP interaction and a targeted point mutation (F309S) improved secretion of FVIII up to 3-fold (Swaroop et al, J Biol Chem 1997). Additional structure and function comparative studies between FV and FVIII may identify new targets for bioengineering strategies with an aim to further enhance the expression of rFVIII in mammalian cell lines. Disclosures:No relevant conflicts of interest to declare.

BiP Binding to the ER-Stress Sensor Ire1 Tunes the Homeostatic Behavior of the Unfolded Protein Response

Author SummarySecreted and membrane-spanning proteins constitute one of every three proteins produced by a eukaryotic cell. Many of these proteins initially fold and assemble in the endoplasmic reticulum (ER). A variety of physiological and environmental conditions can increase the demands on the ER, overwhelming the ER protein folding machinery. To restore homeostasis in response to ER stress, cells activate an intracellular signaling pathway called the unfolded protein response (UPR) that adjusts the folding capacity of the ER according to need. Its failure impairs cell viability and has been implicated in numerous disease states. In this study, we quantitatively interrogate the homeostatic capacity of the UPR. We arrive at a mechanistic model for how the ER stress sensor Ire1 cooperates with its binding partner BiP, a highly redundant ER chaperone, to fine-tune UPR activity. Moving between a predictive computational model and experiments, we show that BiP release from Ire1 is not the switch that activates Ire1; rather, BiP modulates Ire1 activation and deactivation dynamics. BiP binding to Ire1 and its dissociation in an ER stress-dependent manner buffers the system against mild stresses. Furthermore, BiP binding accelerates Ire1 deactivation when stress is removed. We conclude that BiP binding to Ire1 serves to fine-tune the dynamic behavior of the UPR by modulating its sensitivity and shutoff kinetics. This function of the interaction between Ire1 and BiP may be a general paradigm for other systems in which oligomer formation and disassembly must be finely regulated.

Decreased secretion and unfolded protein response up‐regulation are correlated with intracellular retention for single‐chain antibody variants produced in yeast

Heterologous protein expression can easily overwhelm a cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. In the current model of the UPR, the chaperone BiP modulates the activation of the UPR due to its interactions with the signaling protein Ire1p and newly synthesized proteins. In this research, 4-4-20 scFv variants were generated by rational design to alter BiP binding to newly synthesized scFv proteins or via directed evolution aimed at improved secretion. Interestingly, the predicted BiP binding ability did not correlate significantly with the UPR. However, pulse-chase analysis of scFv fate revealed that mutants with a decreased ER residence time were more highly secreted, indicating that improved protein folding was more likely the cause for improved secretion. In fact, decreased secretion correlated with increased binding by BiP, as determined by co-immune precipitation studies. This suggests that the algorithm is not useful for in vivo prediction of variants, and that in vivo screens are more effective for finding variants with improved properties.

The Degree of Folding Instability of the Envelope Protein of a Neurovirulent Murine Retrovirus Correlates with the Severity of the Neurological Disease

A small group of ecotropic murine retroviruses cause a spongiform neurodegenerative disease manifested by tremor, paralysis, and wasting. The neurovirulence of these viruses has long been known to be determined by the sequence of the viral envelope protein, although the nature of the neurotoxicity remains to be clarified. Studies on the neurovirulent viruses FrCas(NC) and Moloney murine leukemia virus ts1 indicate that the nascent envelope protein misfolds, is retained in the endoplasmic reticulum (ER), and induces an unfolded protein response. In the present study we constructed a series of viruses with chimeric envelope genes containing segments from virulent and avirulent retroviruses. Each of the viruses studied was highly neuroinvasive but differed in the severity of the neurological disease they induced. Only viruses that contained the receptor-binding domain (RBD) of the neurovirulent virus induced neurological disease. Likewise, only viruses containing the RBD of the neurovirulent virus exhibited increased binding of the ER chaperone BiP to the envelope precursor protein and induced the unfolded protein response. Thus, the RBD determined both neurovirulence and folding instability. Among viruses carrying the neurovirulent RBD, the severity of the disease was increased when envelope sequences from the neurovirulent virus outside the RBD were also present. Interestingly, these sequences appeared to further increase the degree of folding instability (BiP binding) of the viral envelope protein. These results provide strong support for the hypothesis that this spongiform neurodegenerative disease represents a virus-induced protein folding disorder.

Mechanisms Targeting Apolipoprotein B100 to Proteasomal Degradation

In lipid-poor states, the ubiquitin-proteasomal pathway rapidly degrades misfolded apolipoprotein B100 (apoB) cotranslationally, although the mechanism of delivery from the ER to cytosolic proteasomes is poorly understood. Here we demonstrate key roles of BiP, an ER luminal chaperone, and p97, a cytosolic ATPase anchored to the ER membrane, in the targeting of apoB for proteasomal degradation. Using coimmunoprecipitations, we observed associations of apoB with BiP, p97, Derlin-1, VIMP, and the E3 ubiquitin ligase Hrd1 in HepG2 cells. BiP and p97 were found to bind apoB cotranslationally. Expression of C-terminal truncated apoB molecules in COS-7 cells showed an N-terminal region outside apoB15 and a C-terminal region found in apoB72 were required for BiP and p97 binding, respectively. Interestingly, overexpression of dominant negative p97 demonstrated that the ATPase activity of p97 was essential for proteasomal degradation of apoB but not for apoB binding. However, p97 activity did not appear to affect the N terminus of apoB, which may be cleaved before degradation. These data suggest that p97 and BiP play critical roles in the cotranslational delivery of apoB to proteasomes and formation of a degradative complex. Proteasomal degradation appears to selectively target apoB molecules with large C-terminal domains.

Proteomic Analysis of the alphaIIbbeta3 Interactome Reveals Novel Chaperone and Trafficking Proteins, Including An HSP40 Chaperone, DNAJC10, That Regulates alphaIIbbeta3 Surface Expression

Platelet alpha granules, which contain both membrane-bound and secreted proteins, are formed in the megakaryocyte and then delivered along proplatelet elaborations to the newly forming platelets. A critical but poorly understood process is the post-translational processing, sorting and delivery of proteins to alpha granules prior to their delivery to the proplatelet. Defects in the processing and trafficking of platelet proteins can result in reduced platelet formation. In addition, platelet alpha granules may be differentially packaged with pro- or anti-angiogenic proteins, suggesting that protein sorting events that occur before alpha granule formation may significantly impact platelet end-point function. Thus, research at the intersection of protein trafficking and thrombopoiesis may lead to clearer understanding of the mechanisms of proplatelet formation, the mechanisms of disease in inherited platelet disorders, and the mechanisms of platelet function in inflammation, tumor metastasis and angiogenesis. We have used the platelet integrin aIIbb3 as a model protein to investigate the mechanisms of these processes. In this study we identified proteins that interacted with aIIb in either HEK293 cells or stem cell derived megakaryocytes. The purpose of expanding the “interactome” of aIIb is to identify novel protein-protein interactions that are important for protein delivery to the megakaryocyte surface, and thus could be important for alpha granule and proplatelet formation. We used two methods of capturing interacting proteins:1) a two-cell pull-down assay using Histidine-tagged aIIb and b3 as bait for umbilical cord blood derived megakaryocyte lysate, followed by nickel bead extraction,2) a crosslinking assay in which photoreactive, crosslinking amino acids are incorporated into growing megakaryocytes, then crosslinked by exposure to UV light. To enrich the population of aIIb residing in the ER and Golgi, a mutant aIIb subunit containing a R858G mutation, which prevents cleavage of pro-aIIb to mature aIIb resulting in intracellular retention, was used as bait in some assays. The captured proteins from both methods were separated by SDS-PAGE and analyzed by mass spectroscopy. Two or more unique peptides were identified for 93 proteins, and 33 proteins were identified in two or more separate experiments. Of these proteins, 45 were potential protein-trafficking proteins, known to interact with aIIb, or of unknown function. Further analysis of one of these proteins, DNAJC10, suggested that it plays a role in aIIbb3 biogenesis and trafficking. DNAJC10 is an HSP40 type protein with a BiP binding domain and a second domain containing two disulfide isomerase motifs. It has been shown to be induced during ER stress, and may assist in delivering misfolded ER proteins to the proteasome for degradation. Immunoprecipitation of aIIb and b3 followed by immunoblot with anti-DNAJC10 mAb revealed protein bands corresponding to the molecular mass of DNAJC10, indicating direct or indirect physical interaction of aIIb and b3 with DNAJC10. siRNA mediated knockdown of DNAJC10 increased aIIbb3 surface expression on human megakaryocytes by 12%. Together these findings indicate that DNAJC10 interacts with aIIbb3 and may play a role in regulating aIIbb3 surface expression. This study offers new insights into the control of aIIbb3 surface expression and further studies may reveal new targets for anti- or pro-integrin therapies.

Modified expression of coagulation factor VIII by addition of a glycosylation site at the N terminus of the protein

Recently, it was shown that glycoproteins with N-glycans close to the NH(2) terminus can directly enter the calnexin/calreticulin cycle and bypass BiP binding. This should allow efficient secretion of glycoproteins such as factor VIII (FVIII) whose secretion is negatively affected by BiP interaction. Examination of the glycosylation pattern of the NH(2) terminus of FV and FVIII revealed N-glycans at positions 23 and 27 in FV and at position 41 in FVIII. To improve FVIII secretion, a 14-amino-acid-long polypeptide with (G3) or without (G0; control) three N-linked glycosylation consensus sites was inserted upstream of the NH(2) terminus of a B-domain deleted FVIII protein. Expression of G3- and G0-constructs in three different cell lines resulted in the same or even higher expression rate of protein as found for the B-domain deleted FVIII. However, as demonstrated by Western blot analysis, the G3- as well as the G0-protein variants were mainly retained inside the cells in similar amounts. Thus, glycosylation alone does not automatically lead to higher secretion rates, but must be in context to the normal structure of the FVIII protein.

The Role of BiP in Endoplasmic Reticulum-associated Degradation of Major Histocompatibility Complex Class I Heavy Chain Induced by Cytomegalovirus Proteins

Human cytomegalovirus (HCMV1) US11 and US2 proteins cause rapid degradation of major histocompatibility complex (MHC) molecules, apparently by ligating cellular endoplasmic reticulum (ER)-associated degradation machinery. Here, we show that US11 and US2 bind the ER chaperone BiP. Four related HCMV proteins, US3, US7, US9, and US10, which do not promote degradation of MHC proteins, did not bind BiP. Silencing BiP reduced US11- and US2-mediated degradation of MHC class I heavy chain (HC) without altering the synthesis or translocation of HC into the ER or the stability of HC in the absence of US11 or US2. Induction of the unfolded protein response (UPR) did not affect US11-mediated HC degradation and could not explain the stabilization of HC when BiP was silenced. Unlike in yeast, BiP did not act by maintaining substrates in a retrotranslocation-competent form. Our studies go beyond previous observations in mammalian cells correlating BiP release with degradation, demonstrating that BiP is functionally required for US2- and US11-mediated HC degradation. Further, US2 and US11 bound BiP even when HC was absent and degradation of US2 depended on HC. These data were consistent with a model in which US2 and US11 bridge HC onto BiP promoting interactions with other ER-associated degradation proteins.

Retention of Misfolded Mutant Transthyretin by the Chaperone BiP/GRP78 Mitigates Amyloidogenesis

Carriers of the D18G transthyretin (TTR) mutation display an unusual central nervous system (CNS) phenotype with late onset of disease. D18G TTR is monomeric and highly prone to misfold and aggregate even at physiological conditions. Extremely low levels of mutant protein circulate both in human serum and cerebrospinal fluid, indicating impaired secretion of D18G TTR. Recent data show efficient selective ER-associated degradation (ERAD) of D18G TTR. One essential component of the ER-assisted folding machinery is the molecular chaperone BiP. Co-expression of BiP and D18G TTR, or BiP and wild-type (wt) TTR, or mutants A25T TTR and L55P TTR in Escherichia coli showed that only D18G TTR was significantly captured by BiP. Negligible capture of wt TTR and L55P TTR was seen and a sixfold smaller amount of A25T TTR bound to BiP compared to D18G TTR. These data correlate very well with thermodynamic and kinetic stability of the TTR variants, indicating that folding efficiency is inversely correlated to BiP capture. The complexes between BiP and D18G TTR were stable and could be isolated through affinity chromatography. Analytical ultracentrifugation and size-exclusion chromatography revealed that D18G TTR and BiP formed a mixture of 1:1 complexes and large soluble oligomers. The stoichiometry of captured D18G TTR versus BiP increased with increasing size of the oligomers. This indicates that BiP either worked as a molecular shepherd collecting the aggregation-prone mutant into stable oligomers or that BiP could bind to oligomers formed from misfolded mutant protein. Sequence analysis of bound TTR peptides to BiP revealed a bound sequence corresponding to residues 88-103 of TTR, comprising beta-strand F in the folded TTR monomer constituting part of the hydrogen bonding tetramer interface in native TTR. The F-strand has also been suggested as a possible elongation region of amyloid fibrils, implicating how substoichiomeric amounts of BiP could sequester prefibrillar amyloidogenic oligomers through binding to this part of TTR. BiP binding to D18G TTR was abolished by addition of ATP. The released D18G TTR completely misfolded into amyloid aggregates as shown by ThT fluorescence and Congo red binding.

Structural Analysis of Isoform-Specific Inhibitors Targeting the Tetrahydrobiopterin Binding Site of Human Nitric Oxide Synthases

Nitric oxide synthesized from l-arginine by nitric oxide synthase isoforms (NOS-I-III) is physiologically important but also can be deleterious when overproduced. Selective NOS inhibitors are of clinical interest, given their differing pathophysiological roles. Here we describe our approach to target the unique NOS (6R,1'R,2'S)-5,6,7,8-tetrahydrobiopterin (H(4)Bip) binding site. By a combination of ligand- and structure-based design, the structure-activity relationship (SAR) for a focused set of 41 pteridine analogues on four scaffolds was developed, revealing selective NOS-I inhibitors. The X-ray crystal structure of rat NOS-I dimeric-oxygenase domain with H(4)Bip and l-arginine was determined and used for human isoform homology modeling. All available NOS structural information was subjected to comparative analysis of favorable protein-ligand interactions using the GRID/concensus principal component analysis (CPCA) approach to identify the isoform-specific interaction site. Our interpretation, based on protein structures, is in good agreement with the ligand SAR and thus permits the rational design of next-generation inhibitors targeting the H(4)Bip binding site with enhanced isoform selectivity for therapeutics in pathology with NO overproduction.

The Cotranslational Maturation of the Type I Membrane Glycoprotein Tyrosinase: The Heat Shock Protein 70 System Hands Off to the Lectin-based Chaperone System

The maturation of eukaryotic secretory cargo initiates cotranslationally and cotranslocationally as the polypeptide chain emerges into the endoplasmic reticulum lumen. Here, we characterized the cotranslational maturation pathway for the human type I membrane glycoprotein tyrosinase. To recapitulate the cotranslational events, including glycosylation, signal sequence cleavage, chaperone binding, and oxidation, abbreviated transcripts lacking a stop codon were in vitro translated in the presence of semipermeabilized melanocyte membranes. This created a series of ribosome/translocon-arrested chains of increasing lengths, simulating intermediates in the cotranslational folding process. Initially, nascent chains were found to associate with the heat shock protein (Hsp) 70 family member BiP. As the nascent chains elongated and additional glycans were transferred, BiP binding rapidly decreased and the lectin-based chaperone system was recruited in its place. The lectin chaperone calnexin bound to the nascent chain after the addition of two glycans, and calreticulin association followed upon the addition of a third. The glycan-specific oxidoreductase ERp57 was cross-linked to tyrosinase when calnexin and calreticulin were associated. This timing coincided with the formation of disulfide bonds within tyrosinase and the cleavage of its signal sequence. Therefore, tyrosinase maturation initiates cotranslationally with the Hsp70 system and is handed off to the lectin chaperone system that first uses calnexin before calreticulin. Interestingly, divergence in the maturation pathways of wild-type and mutant albino tyrosinase can already be observed for translocon-arrested nascent chains.

Stable Binding of ATF6 to BiP in the Endoplasmic Reticulum Stress Response

Endoplasmic reticulum (ER) stress-induced activation of ATF6, an ER membrane-bound transcription factor, requires a dissociation step from its inhibitory regulator, BiP. It has been generally postulated that dissociation of the BiP-ATF6 complex is a result of the competitive binding of misfolded proteins generated during ER stress. Here we present evidence against this model and for an active regulatory mechanism for dissociation of the complex. Contradictory to the competition model that is based on dynamic binding of BiP to ATF6, our data reveal relatively stable binding. First, the complex was easily isolated, in contrast to many chaperone complexes that require chemical cross-linking. Second, ATF6 bound at similar levels to wild-type BiP and a BiP mutant form that binds substrates stably because of a defect in its ATPase activity. Third, ER stress specifically induced the dissociation of BiP from ER stress transducers while the competition model would predict dissociation from any specific substrate. Fourth, the ATF6-BiP complex was resistant to ATP-induced dissociation in vitro when isolated without detergents, suggesting that cofactors stabilize the complex. In favor of an active dissociation model, one specific region within the ATF6 lumenal domain was identified as a specific ER stress-responsive sequence required for ER stress-triggered BiP release. Together, our results do not support a model in which competitive binding of misfolded proteins causes dissociation of the BiP-ATF6 complex in stressed cells. We propose that stable BiP binding is essential for ATF6 regulation and that ER stress dissociates BiP from ATF6 by actively restarting the BiP ATPase cycle.

Endoplasmic Reticulum (ER) Stress Induced by a Neurovirulent Mouse Retrovirus Is Associated with Prolonged BiP Binding and Retention of a Viral Protein in the ER

Some murine retroviruses cause a spongiform neurodegenerative disease exhibiting pathology resembling that observed in transmissible spongiform encephalopathies. The neurovirulence of these "spongiogenic retroviruses" is determined by the sequence of their respective envelope proteins, although the mechanisms of neurotoxicity are not understood. We have studied a highly neurovirulent virus called FrCasE that causes a rapidly progressive form of this disease. Recently, transcriptional markers of endoplasmic reticulum (ER) stress were detected during the early preclinical period in the brains of FrCasE-infected mice. In contrast, ER stress was not observed in mice infected with an avirulent virus, F43, which carries a different envelope gene, suggesting a role for ER stress in disease pathogenesis. Here we have examined in NIH 3T3 cells the cause of this cellular stress response. The envelope protein of F43 bound BiP, a major ER chaperone, transiently and was processed normally through the secretory pathway. In contrast, the envelope protein of FrCasE bound to BiP for a prolonged period, was retained in the ER, and was degraded by the proteasome. Furthermore, engagement of the FrCasE envelope protein by ER quality control pathways resulted in decreased steady-state levels of this protein, relative to that of F43, both in NIH 3T3 cells and in the brains of infected mice. Thus, the ER stress induced by FrCasE appears to be initiated by inefficient folding of its viral envelope protein, suggesting that the neurodegenerative disease caused by this virus represents a protein misfolding disorder.

A phaseolin domain involved directly in trimer assembly is a determinant for binding by the chaperone BiP.

The binding protein (BiP; a member of the heat-shock 70 family) is a major chaperone of the endoplasmic reticulum (ER). Interactions with BiP are believed to inhibit unproductive aggregation of newly synthesized secretory proteins during folding and assembly. In vitro, BiP has a preference for peptide sequences enriched in hydrophobic amino acids, which are expected to be exposed only in folding and assembly intermediates or in defective proteins. However, direct information regarding sequences recognized in vivo by BiP on real proteins is very limited. We have shown previously that newly synthesized monomers of the homotrimeric storage protein phaseolin associate with BiP and that phaseolin trimerization in the ER abolishes such interactions. Using different phaseolin constructs and green fluorescent protein (GFP) fusion proteins, we show here that one of the two alpha-helical regions of polypeptide contact in phaseolin trimers (35 amino acids located close to the C terminus and containing three potential BiP binding sites) effectively promotes BiP association with phaseolin and with secretory GFP fusions expressed in transgenic tobacco or in transfected protoplasts. We also show that overexpressed BiP transiently sequesters phaseolin polypeptides. We conclude that one of the regions of monomer contact is a BiP binding determinant and suggest that during the synthesis of phaseolin, the association with BiP and trimer formation are competing events. Finally, we show that the other, internal region of contact between monomers is necessary for phaseolin assembly in vivo and contains one potential BiP binding site.

The function of GADD34 is a recovery from a shutoff of protein synthesis induced by ER stress: elucidation by GADD34-deficient mice.

GADD34 is a protein that is induced by stresses such as DNA damage. The function of mammalian GADD34 has been proposed by in vitro transfection, but its function in vivo has not yet been elucidated. Here we generated and analyzed GADD34 knockout mice. Despite their embryonic stage- and tissue-specific expressions, GADD34 knockout mice showed no abnormalities at fetal development and in early adult life. However, in GADD34-/- mouse embryonic fibroblasts (MEFs), recovery from a shutoff of protein synthesis was delayed when MEFs were exposed to endoplasmic reticulum (ER) stress. The phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2alpha) at Ser51 induced by thapsigargin or DTT was prolonged in GADD34-/- MEF, although following treatment with tunicamycin, the eIF2alpha phosphorylation level did not change in either GADD34+/+ or GADD34-/- cells. ER stress stimuli induced expressions of Bip (binding Ig protein) and CHOP (C/EBP homologous protein) in MEF of wild-type mice. These expressions were strongly reduced in GADD34-/- MEF, which suggests that GADD34 up-regulates Bip and CHOP. These results indicate that GADD34 works as a sensor of ER stress stimuli and recovers cells from shutoff of protein synthesis.

Amino acids in the TM4-TM5 loop of Na,K-ATPase are important for biosynthesis.

The ten-transmembrane Na,K-ATPase alpha-subunit exposes very few amino acids to the extra membrane space except for an approximately 408 residue-long loop between transmembrane segments four and five. The present paper focuses on the role of this loop in biosynthesis of functional Na,K-ATPase. Expression of 39 mutations in this loop to phylogenetically conserved as well as nonconserved residues showed that only two could be expressed at 30 degrees C. By contrast, only five could not be produced in a functional form at 15 degrees C. A detailed analysis showed that a number of these mutants are temperature-sensitive folding mutants, as they induce the unfolded protein response at 30 degrees C but not at 15 degrees C. We used an algorithm to predict that residues (868)ENGFLIPIHLL(878) in the L78 loop exposed to the endoplasmic reticulum lumen constitute the most likely BiP binding site. Correct folding of this sequence may be important in the endoplasmic reticulum quality control, as the same loop is responsible for the alpha-beta-associations required to leave this compartment. On the basis of the Ca-ATPase crystal structure and the presented data, we propose a model to account for the role of the TM4-TM5 loop in Na,K-ATPase biosynthesis.

UDP-Glc:glycoprotein glucosyltransferase recognizes structured and solvent accessible hydrophobic patches in molten globule-like folding intermediates.

Protein folding in the cell involves the action of different molecular chaperones and folding-facilitating enzymes. In the endoplasmic reticulum (ER), the folding status of glycoproteins is stringently controlled by a glucosyltranferase enzyme (GT) that creates monoglucosylated structures recognized by ER resident lectins (calnexincalreticulin, CNXCRT). GT serves as a folding sensor because it only glucosylates misfolded or partly folded glycoproteins. Nevertheless, the molecular mechanism behind this recognition process remains largely unknown. In this paper we explore the structural determinants for GT recognition by using a single domain model protein. For this purpose we used a family of chemically glycosylated proteins derived from chymotrypsin inhibitor-2 as GT substrates. Structural characterization of species showing higher glucose acceptor capacity suggests that GT recognizes solvent accessible hydrophobic patches in molten globule-like conformers mimicking intermediate folding stages of nascent glycoproteins. It was further confirmed that BiP (binding protein, a chaperone of the heat shock protein 70 family) preferentially recognized neoglycoproteins displaying extended conformations, thus providing a molecular rationale for the sequential BiP-CNXCRT interaction with folding glycoproteins observed in vivo.

ER Stress Regulation of ATF6 Localization by Dissociation of BiP/GRP78 Binding and Unmasking of Golgi Localization Signals

ATF6 is an endoplasmic reticulum (ER) stress-regulated transmembrane transcription factor that activates the transcription of ER molecular chaperones. Upon ER stress, ATF6 translocates from the ER to the Golgi where it is processed to its active form. We have found that the ER chaperone BiP/GRP78 binds ATF6 and dissociates in response to ER stress. Loss of BiP binding correlates with the translocation of ATF6 to the Golgi, which was slowed in cells overexpressing BiP. Two Golgi localization signals (GLSs) were identified in ATF6. Removal of BiP binding sites from ATF6, while retaining a GLS, resulted in its constitutive translocation to the Golgi. These results suggest that BiP retains ATF6 in the ER by inhibiting its GLSs and that dissociation of BiP during ER stress allows ATF6 to be transported to the Golgi.

Structural requirements for inhibition of the neuronal nitric oxide synthase (NOS-I): 3D-QSAR analysis of 4-oxo- and 4-amino-pteridine-based inhibitors.

The family of homodimeric nitric oxide synthases (NOS I-III) catalyzes the generation of the cellular messenger nitric oxide (NO) by oxidation of the substrate L-arginine. The rational design of specific NOS inhibitors is of therapeutic interest in regulating pathological NO levels associated with sepsis, inflammatory, and neurodegenerative diseases. The cofactor (6R)-5,6,7,8-tetrahydrobiopterin (H(4)Bip) maximally activates all NOSs and stabilizes enzyme quaternary structure by promoting and stabilizing dimerization. Here, we describe the synthesis and three-dimensional (3D) quantitative structure-activity relationship (QSAR) analysis of 65 novel 4-amino- and 4-oxo-pteridines (antipterins) as inhibitors targeting the H(4)Bip binding site of the neuronal NOS isoform (NOS-I). The experimental binding modes for two inhibitors complexed with the related endothelial NO synthase (NOS-III) reveal requirements of biological affinity and form the basis for ligand alignment. Different alignment rules were derived by building other compounds accordingly using manual superposition or a genetic algorithm for flexible superposition. Those alignments led to 3D-QSAR models (comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA)), which were validated using leave-one-out cross-validation, multiple analyses with two and five randomly chosen cross-validation groups, perturbation of biological activities by randomization or progressive scrambling, and external prediction. An iterative realignment procedure based on rigid field fit was used to improve the consistency of the resulting partial least squares models. This led to consistent and highly predictive 3D-QSAR models with good correlation coefficients for both CoMFA and CoMSIA, which correspond to experimentally determined NOS-II and -III H(4)Bip binding site topologies as well as to the NOS-I homology model binding site in terms of steric, electrostatic, and hydrophobic complementarity. These models provide clear guidelines and accurate activity predictions for novel NOS-I inhibitors.

Specificity of Peptide-Induced Depolymerization of the Recombinant Carboxy-Terminal Fragment of BiP/GRP78

Inthe present study, we have used a non-denaturing gel electrophoresis assay to characterize the specificity of the peptide-induced depolymerization process of the isolated recombinant C-terminal domain (C30) of the molecular chaperone BiP, in the presence of specific synthetic peptides and with the neuropeptide Substance P. In the absence of peptidic ligand, C30 self-associates readily into multiple oligomeric species. Upon peptide addition, C30 oligomers convert into dimers, then into monomers. Our data indicate that the algorithm we previously developed to predict putative BiP binding sites in any protein sequence is also a good indicator as to whether a peptide can efficiently induce depolymerization of the C-terminal peptide binding domain and stimulate the ATPase activity of the full-length protein.